Clinical manufacture of therapeutic proteins is an expensive, large scale endeavor. Maintaining cell growth and viability throughout the cell culture process is very important. As the demand for greater quantities of therapeutic recombinant proteins increases, positive increases in protein production, cell growth and viability are sought out by implementing new methods to improve cell development, media optimization and process control parameters, and to intensify harvest and purification processes. Much effort is now being placed on process optimization, particularly methods and formulations for feeding production cell cultures. One such method is the use of a concentrated feed medium during the production phase, often used in fed batch culture processes, to improve protein titer, cell growth, and/or cell viability.
Tyrosine is a critical amino acid for sustaining cultured cells in growth and viability and is included in growth media and concentrated production media formulations. A depletion of tyrosine in a production culture can lead to decreases in cell growth, viability and/or protein production.
Due to poor solubility at neutral pH, tyrosine cannot be compounded at high concentrations in either the growth or concentrated feed medium. Therefore, only a limited amount of tyrosine can be compounded into media formulations without causing the media to precipitate. Phenylalanine can convert to tyrosine via the enzyme phenylalanine hydroxylase (PAH) in certain cell types and can also be included in media formulations.
Cysteine is another important amino acid for sustaining cultured mammalian cells. However, cysteine readily oxidizes to form cystine in neutral or slightly alkaline solutions. So while cysteine is freely soluble in water, it may contribute to insolubility and/or precipitation when in its oxidized form.
Mammalian cells are typically grown in cultures that are at or near neutral pH, such as from about pH 6.5 to about pH 7.5. Tyrosine and cysteine, necessary components of mammalian cell culture media, can cause media formulations to destabilize in the neutral conditions necessary for cell growth.
New cell culture media formulations and/or methods for feeding production cultures that provide even incremental improvements in cell growth, viability and/or protein production are valuable, given the expense of large scale cell culture processes and the difficulty and expense of building and obtaining regulatory approval for new large-scale, commercial culture facilities.
There is a continuing need to develop media formulations and feeding methods that are able to provide adequate amino acid levels in a production culture to maintain and improve cell viability, specific productivity, and titer. Any improvements to cell culture media formulations and/or feeding strategies that allow for flexibility and customization to facilitate desirable recombinant polypeptide expression, titer, cell growth and/or cell viability can lead to ease of maintenance, higher production levels, thereby reducing the costs associated with manufacturing protein therapeutics. The invention fulfills these needs by providing a simple, easy and inexpensive method of increasing cell growth and protein production.